Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.
Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.
Duong
F
F
Dartmouth Medical School, Department of Biochemistry, 7200 Vail Building, Hanover, NH 03755, USA.
Wickner
W
W
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D001692:Biological Transport / Q000502:physiology; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; C057184:outer membrane protein A precursor (E coli); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins